Surface PD-1 expression in T cells is suppressed by HNRNPK through an exonic splicing silencer on exon 3.

OBJECTIVE: Immunotherapy targeting programmed cell death 1 (PDCD1 or PD-1) and its ligands has shown remarkable promise and the regulation mechanism of PD-1 expression has received arising attention in recent years. PDCD1 exon 3 encodes the transmembrane domain and the deletion of exon 3 produces a soluble protein isoform of PD-1 (sPD-1), which can enhance immune response by competing with full-length PD-1 protein (flPD-1 or surface PD-1) on T cell surface. However, the mechanism of PDCD1 exon 3 skipping is unclear. METHODS: The online SpliceAid program and minigene expression system were used to analyze potential splicing factors involved in the splicing event of PDCD1 exon 3. The potential binding motifs of heterogeneous nuclear ribonucleoprotein K (HNRNPK) on exon 3 predicted by SpliceAid were mutated by site-directed mutagenesis technology, which were further verified by pulldown assay. Antisense oligonucleotides (ASOs) targeting the exonic splicing silencer (ESS) on PDCD1 exon 3 were synthesized and screened to suppress the skipping of exon 3. The alternative splicing of PDCD1 exon 3 was analyzed by semiquantitative reverse transcription PCR. Western blot and flow cytometry were performed to detect the surface PD-1 expression in T cells. RESULTS: HNRNPK was screened as a key splicing factor that promoted PDCD1 exon 3 skipping, causing a decrease in flPD-1 expression on T cell membrane and an increase in sPD-1 expression. Mechanically, a key ESS has been identified on exon 3 and can be bound by HNRNPK protein to promote exon 3 skipping. Blocking the interaction between ESS and HNRNPK with an ASO significantly reduced exon 3 skipping. Importantly, HNRNPK can promote exon 3 skipping of mouse Pdcd1 gene as well. CONCLUSIONS: Our study revealed a novel evolutionarily conserved regulatory mechanism of PD-1 expression. The splicing factor HNRNPK markedly promoted PDCD1 exon 3 skipping by binding to the ESS on PDCD1 exon 3, resulting in decreased expression of flPD-1 and increased expression of sPD-1 in T cells.

An anti-PD-1 antisense oligonucleotide promotes the expression of soluble PD-1 by blocking the interaction between SRSF3 and an exonic splicing enhancer of PD-1 exon 3.

PD-1 is a key immune checkpoint molecule. Anti-PD-1 immunotherapy is encouraging in cancer treatment. However, it still needs to be improved. PD-1 has at least five isoforms generated by alternative splicing. An isoform without exon 3 encoding soluble PD-1 (sPD-1) can activate anti-tumor immunity by inhibiting the interaction between cellular surface full-length PD-1 (flPD-1) and PD-L1. However, the regulatory mechanism of exon 3 splicing remains largely unknown. Here, we screened the exon 3 sequence by mutation and searched corresponding splicing factors by SpliceAid database and pulldown assay. The alternative splicing of PD-1 exon 3 was analyzed by RT-PCR. The expression levels of flPD-1 and sPD-1 were analyzed by Western blot, flow cytometry, and ELISA. We discovered that an exonic splicing enhancer (ESE) of exon 3 is essential for its inclusion. Moreover, SRSF3 can bind to this ESE and enhance exon 3 inclusion and flPD-1 expression. We designed and screened out an antisense oligonucleotide (ASO) targeting PD-1 to block the interaction between SRSF3 and ESE, and significantly increase exon 3 skipping and sPD-1 expression, which was verified in various tumor cells in addition to oral cancer cells. Altogether, our results uncovered the regulatory mechanism of human PD-1 exon 3 splicing and sPD-1 expression and further designed a novel anti-PD-1 ASO, which are useful for developing a new method of anti-cancer immunotherapy.

The Tibetan Antelope Population Depends on Lakes on the Tibetan Plateau.

The influence of freshwater ecosystems on terrestrial taxa in high-altitude regions with challenging access, such as the Tibetan Plateau, remains inadequately understood. This knowledge gap is particularly significant due to the fragility of these ecosystems, characterized by low primary productivity. Ungulates, in particular, may exhibit high sensitivity to even minor alterations in plant availability, potentially stemming from global climate change. Consequently, the investigation of these ecosystems may offer valuable insights into addressing future challenges posed by climate change. Here, to fill this knowledge gap, we explore the relationship between lakes and Tibetan antelopes in an even more vulnerable region, the Tibetan Plateau. We found that the Tibetan antelope population was higher in areas with larger lakes, and where the terrain near the lakes was flatter. At the same time, vegetation cover and plant diversity were higher near the lake compared to areas farther away from the lake. This phenomenon can be elucidated by the fact that lakes offer Tibetan antelopes a richer food supply and reduced predation risk. Our study provides new perspectives for researchers to explore the cross-ecosystem impacts of climate change.

HnRNP A1 Suppresses the Odontogenic Differentiation and the Inclusion of RUNX2 Exon 5 of Dental Mesenchymal Cells.

BACKGROUND: RUNX2 (Runt-related transcription factor 2) acts as a key regulator in the odontogenic differentiation of human dental pulp stem cells (hDPSCs). Moreover, the inclusion of exon 5 is important for RUNX2 function. Our previous study showed that Y-Box Binding Protein 1 (YBX1) promoted RUNX2 exon 5 inclusion and mineralization of hDPSCs. However, the regulatory mechanism of RUNX2 exon 5 alternative splicing needed further exploration. METHODS: The expression level of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) during the odontogenic differentiation of hDPSCs was analyzed by RT-PCR and Western blot. The roles of hnRNP A1 in the alternative splicing of RUNX2 exon 5 and the odontogenic differentiation of dental mesenchymal cells were analyzed by gain- and loss-of-function experiments. RESULTS: Surprisingly, we found an alternative splicing factor, hnRNP A1, which had an opposite role to YBX1 in regulating RUNX2 exon 5 inclusion and odontogenic differentiation of hDPSCs. Through gain- and loss-of-function assay, we found that hnRNP A1 suppressed the inclusion of RUNX2 exon 5, resulting in the inhibition of odontoblastic differentiation. The overexpression of hnRNP A1 can inhibit the expression of ALP (alkaline phosphatase) and OCN (osteocalcin), and the formation of mineralized nodules during the odontogenic differentiation of both hDPSCs and mouse dental papilla cells (mDPCs), whereas the opposite results were obtained with an hnRNP A1 knockdown preparation. CONCLUSIONS: The present study indicated that hnRNP A1 suppressed RUNX2 exon 5 inclusion and reduced the odontogenic differentiation ability of hDPSCs and mDPCs.

PD-L1 Exon 3 Is a Hidden Switch of Its Expression and Function in Oral Cancer Cells.

The interaction between programmed cell death 1 ligand 1 (PD-L1) and programmed cell death protein 1 (PD-1) protects tumor cells from immune surveillance. PD-L1 exon 3 is a potential alternative exon and encodes an Ig variable (IgV) domain. Here, we found that a lack of exon 3 leads to the significant loss of cellular membrane locations and the dramatically reduced protein expression of PD-L1, indicating that PD-L1 exon 3 is essential for its protein expression and translocation to the cell membrane. Notably, oral cancer cells show almost no exon 3 skipping to ensure the expression of the full-length, functional PD-L1 protein. We discovered two key exonic splicing enhancers (ESEs) for exon 3 inclusion. Two efficient antisense oligonucleotides (ASOs) were identified to block these two ESEs, which can significantly trigger exon 3 skipping and decrease the production of full-length, functional PD-L1 on the surface of cancer cells. Treatment of oral cancer cells with these ASOs significantly enhanced immune cells' suppression of cancer cell proliferation. Surprisingly, these two ASOs also significantly inhibited cell growth and induced cell pyroptosis in oral cancer cells. Altogether, the results of our study demonstrate the pivotal roles of exon 3 in PD-L1 expression and provide a novel anti-PD-L1 method.

Enhancer RNA IRS2e is essential for IRS2 expression and the oncogenic properties in oral squamous cell carcinoma.

The crucial roles of enhancer RNAs (eRNAs) in the regulation of gene expression in human diseases have drawn wider and wider attention in recent years. However, the specific expression profile and function of eRNAs are still rarely discussed in oral squamous cell carcinoma (OSCC), the most common subtype of head and neck squamous cell carcinoma (HNSC). In this study, we aimed to investigate the expression and function of an uncharacterized eRNA, insulin receptor substrate 2 enhancer RNA (IRS2e), in OSCC. We found that IRS2e was overexpressed in HNSC and its overexpression was positively correlated with a poor prognosis. The downregulation of IRS2e by short hairpin RNA significantly inhibited cell growth and induced cellular apoptosis and cell-cycle arrest in OSCC cells. Furthermore, the ablation of IRS2e inhibited tumor growth in vivo. Mechanically, IRS2e is essential for the expression of insulin receptor substrate 2 (IRS2), an oncogene nearby IRS2e in chromosome 13. Altogether, our study demonstrated that IRS2e is a novel oncogenic eRNA required for oncogene IRS2 expression in OSCC.

Enhancing the immunogenicity of a DNA vaccine against Streptococcus mutans by attenuating the inhibition of endogenous miR-9.

DNA vaccine provides a promising method for preventing and treating diseases. However, the low immunogenicity restricts its application. New approaches are urgent to be explored to enhance the immune response of DNA vaccine. MicroRNAs are endogenous, small non-coding RNAs which play parts in gene expression inhibition. In this study, microRNA-9 (miR-9) was found to inhibit the expression of the GLU-A-P antigen protein encoded by the anti-caries DNA vaccine. Mutation of miR-9 binding sites in the gene fragment encoding GLU-A-P antigen protein significantly increased the expression of antigen protein. Moreover, miR-9 sponge can improve the expression of the GLU-A-P antigen protein. The co-immunization with miR-9 sponge and anti-caries DNA vaccine significantly enhanced the specific immune response in vivo. In conclusion, attenuating the inhibition of endogenous miR-9 enhanced the antigen expression and immunogenicity of the anti-caries DNA vaccine.

Downregulation of SRSF3 by antisense oligonucleotides sensitizes oral squamous cell carcinoma and breast cancer cells to paclitaxel treatment.

PURPOSE: Paclitaxel (PTX) is widely used in the chemotherapy of many cancers, including breast cancer and oral squamous cell carcinoma (OSCC). However, many patients respond poorly to PTX treatment. The SRSF3 oncogene and several splicing factors play important roles in OSCC tumorigenesis. This study aimed to understand the function of splicing factors in PTX treatment and improve the therapeutic effects of PTX treatment. METHODS: Splicing factors regulated by PTX treatment were screened in CAL 27 cell by reverse transcription polymerase chain reaction. The function of SRSF3 in PTX treatment was analyzed by gain-of-function or loss-of-function assay in OSCC cell lines CAL 27 and SCC-9 and breast cancer cell line MCF-7. Alternative splicing of SRSF3 exon 4 in cancer tissues or cells was analyzed by RT-PCR and online program TSVdb. SRSF3-specific antisense oligonucleotide (ASO) SR-3 was used to downregulate SRSF3 expression and enhance the effect of PTX treatment. RESULTS: PTX treatment decreased SRSF3 expression, and SRSF3 overexpression rescued the growth inhibition caused by PTX in both OSCC and breast cancer cells. Moreover, we found that PTX treatment could repress SRSF3 exon 4 (containing an in-frame stop codon) exclusion and then decrease the SRSF3 protein expression. Increased exclusion of SRSF3 exon 4 is correlated with poor survival in OSCC and breast cancer patients. SR-3 downregulated SRSF3 protein expression and significantly increased the sensitivity of cancer cells to PTX treatment. CONCLUSIONS: SRSF3 downregulation by ASO sensitizes cancer cells to PTX treatment.

[Effects on the activities and mRNA expression of CYP3A in rat's liver by four kinds of extracts from anti-cancer Traditional Chinese Medicines].

OBJECTIVE: To study the effects of four kinds of extracts from anti-cancer Taditional Chinese Medicines on the activity and mRNA expression of CYP3A in rat's liver. METHODS: Rat's liver microsomal cytochrome P450 and CYP3A isoemzymes--erythromycin N-demethylase(ERD) activities were determined by UV chromatography, the mRNA expression levels of CYP3Al and CYP3A2 were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Extracts of Rhizoma Curcumae, Rhizoma Atractylodes and Rhizoma Atractylodes Macrocaphalae markedly increased the P450 content of liver microsomes and induced the enzyme activity of CYP3A, but extract of Poria cocos did not. At the mRNA level, extracts of Rhizoma Curcumae, Rhizoma Atractylodes Lanceae and Rhizoma Atractylodes Macraphalae induced the expression of CYP3A1, but extract of Poria cocos did not. The expression of CYP3A2 were induced by extracts of Rhizoma Curcumae and Rhizoma Atractylodes Lanceae, but extracts of Rhizoma Atractylodes Macrocaphalae and Poria cocos were found of remarkable inhibition of the mRNA expression of CYP3A2. CONCLUSION: Extracts of Rhizoma Curcumae, Rhizoma Atractylodes Lanceae and Rhizoma Atractylodes Macrocaphalae can regulate CYP3A on the levels of enzyme activity and mRNA expression, but Poria cocos extract only regulated CYP3A on the level of mRNA expression, possibly by affecting the metabolism of other drugs in the body.